AN INVESTIGATION OF A TEMPERATURE-SENSITIVE INTEIN WITH REGARDS TO ITS USE AS METABOLIC CONTROL ELEMENT FOR BIOPROCESS ENHANCEMENT by Daniel Korvin B.Sc., The University of British Columbia, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2018 © Daniel Korvin, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: An Investigation of a Temperature-Sensitive Intein with regards to its use as a Metabolic Control Element for Bioprocess Enhancement submitted by Daniel Korvin in partial fulfillment of the requirements for the degree of Master of Applied Science in Chemical and Biological Engineering Examining Committee: Dr. Vikramaditya G. Yadav Supervisor Dr. Marko Kryworuchko Supervisory Committee Member Dr. Susan Baldwin Supervisory Committee Member Additional Examiner Additional Supervisory Committee Members: Supervisory Committee Member Supervisory Committee Member ii Abstract Bioprocesses based on metabolically engineered microbes have become tremendously important in recent decades as a platform for the synthesis of complex molecules. Substantial research effort has been devoted to the improvement of microbial strains involved, and while this has enhanced some metrics of strain performance dramatically, namely product yield with respect to substrate and biomass, achieving similar results with other aspects has remained elusive. Improving productivity, the rate at which a modified strain can synthesize a product of interest, in particular has presented an engineering challenge despite its obvious value to the economics of a process and has typically only been done through bioprocess optimization. A strategy that could yield the desired result is strain engineering to better integrate with the bioprocess context in which it is used. The work described in this thesis has sought to achieve that goal by providing a method to the operating engineer to dynamically control the induction of genes associated with product formation. More specifically, a T7 RNA polymerase was modified by the insertion of a mutant variant of the S cerevisiae Vacuolar membrane ATPase (VMA) intein. This mutant intein will only splice out of its host only under conditions of reduced temperature, which in effect makes the polymerase active only after a temperature shift from 37o C to 18o C degrees. This creates a strict demarcation between biomass accumulation and product synthesis, only allowing this transition to be made at an optimal point during fermentation, as chosen by the operating engineer. Using lycopene biosynthesis as a case study and applying this approach, it was found that a productivity improvement of approximately 15% over an uncontrolled strain was attained. It was also found that a remarkable degree of control stringency was conferred upon the system, with no premature product synthesis detected under any condition investigated. These results are expanded upon to generate a series of simple mathematical models, with the aim of describing how such a dynamic metabolic control element might be expected to perform in a more generalized context, and to provide a means by which to more quantitatively assess the strain’s performance. iii Lay Summary In recent decades, the production of fine and commodity chemicals has increasingly been done through the use of metabolically engineered microbes. The aim of this thesis is to engineer such a microbe, and to demonstrate how its productivity can be improved by including a generalizable enzymatic switch that lets the operator turn on the microbe’s ability to produce the chemical lycopene, used here as a test case, when it is most efficient to do so. The input signal required to activate the switch is a drop in temperature from 37o C to 18o C, chosen because it has previously been shown to improve microbe-based lycopene production, and because temperature is a parameter over which the operator has a high level of control. Other inputs could be used to create this improvement, if the system was engineered appropriately, and some of these possibilities are theoretically explored as well. iv Preface This thesis, entitled “An Investigation of a Temperature-Sensitive Intein with regards to its Use as a Metabolic Control Element for Bioprocess Enhancement” presents the research that I performed during the course of my MASc, and was identified, initiated, and supervised by Professor Vikramaditya G. Yadav. A version of part of this study, namely the methods used, and some the data in sections 4.2, 4.3, and 4.4, was published previously (Korvin, Daniel, and Vikramaditya G. Yadav. “A Molecular Switch That Enhances Productivity of Bioprocesses for Heterologous Metabolite Production.” Molecular Systems Design & Engineering, vol. 3, no. 3, 2018, pp. 550–559., doi:10.1039/c8me00013a.). For this paper, I conducted experiments and gathered data. The numerical model was developed, and the analysis performed by Dr. Yadav and I. I prepared the first draft of the paper which was subsequently revised by Dr. Yadav. v Table of Contents Abstract………………………………………………………………………………………..…………..iii Lay Summary…………………………………………………………………………………………...…iv Preface………………………………………………………………………………………………………v Table of Contents……………………………………………………………………….…………..……..vi List of Tables……………………………………………………………………………..……………….vii List of Figures……………………………………………………………………..……………………..viii Chapter 1: Introduction…………………………………………………………………………..……… 1 Chapter 2: Literature Review…………………………………………………………………………..…5 2.1 The Intein……………………………………………………………………………………….5 2.2 The Origin and Function of Inteins………………………..……………………………………8 2.3 Inteins in Biotechnology……………………………………………………………………....10 2.4 The MEP and MVA Isoprenoid Pathways……………………………………………...…….14 2.5 Isoprenoids in Metabolic Engineering……………………………………………………..….19 2.6 Engineering Dynamic Control of Metabolism……………………………………………..….21 Chapter 3: Methods and Materials………………………………………………………………….…..27 3.1 Reagents and Materials………………………………………………………………………..27 3.2 Molecular Cloning and Genetic Engineering…………………………………………………27 3.3 Growth and Activity Assays…………………………………….…………………………….30 Chapter 4: Results and Discussion………………………………………………………………..……..33 4.1 Molecular Biology and DNA Assembly………………………………………………..……..33 4.2 Temperature-Sensitive Intein Validation…………………………………………………..….36 4.3 Dynamically Controlled Lycopene Bioynthesis………………………………………………41 4.4 Predictive Model Development and Analysis………………………………………….……..48 4.5 Fermentation Model Development and Parameterization…………………………………….59 Chapter 5: Conclusion and Future Directions……………………………………………………..….. 67 Bibliography…………………………………………………………………………………………...….71 vi List of Tables Table 4.1 Definitions of parameters and variables in section 4.4 and 4.5……………………..…58 Table 4.2 Parameter values of pRNAP-TS and pRNAP-Dead at 37 °C and 18 °C………….…..66 vii List of Figures Figure 1.1 All T7 RNA polymerase constructs used in this work…………………………………4 Figure 2.1 The mechanism of class 1 intein splicing………………………………………..…….7 Figure 2.2 The MEP and MVA pathways…………………………………………………..……18 Figure 3.1 The metabolic pathway to lycopene biosynthesis……………………………….……30 Figure 4.1 Agarose gel electrophoresis after PCR amplification of DNA fragments used in this work……………………………………………………………………………………..………..35 Figure 4.2 Agarose gel electrophoresis after PCR amplification of DNA fragments used in this work……………………………………………………………………………………...……….35 Figure 4.3 1.5% LB agar plates with each of the experimental E coli strains, grown overnight at 18 and 37 degrees celsius as indicated……………………………………………………...……39 Figure 4.4 Normalized activity of experimental strains at 37 degrees celsius……………..…….39 Figure 4.5 Normalized activity of experimental strains at 18 degrees celsius………………..….40 Figure 4.6 Growth comparison of the 4 experimental strains while coexpressing a lycopene expression gene cassette………………………………………………………………………….45 Figure 4.7 Relative impact of IPTG concentration on product concentration……………………46 Figure 4.8 Product synthesis comparison of the 4 experimental strains while coexpressing a lycopene expression gene cassette……………………………………………………….……….47 Figure 4.9 Dimensionless biomass and product formation ratio of a hypothetical dynamically controlled strain vs a conventional expression strain…………………………………………….55 Figure 4.10 Dimensionless biomass and product formation ratio of a hypothetical dynamically controlled strain vs a conventional expression strain…………………………………………….56 viii Figure 4.11 Dimensionless biomass and product formation ratio of a hypothetical dynamically controlled strain vs a conventional expression strain ………………...…………………...……..57 Figure 4.12 Experimental validation of the impact of induction time on the performance of a strain with product synthesis controlled by a dynamic control element …………………………57 Figure 4.13 Substrate consumption and biomass accumulation of the pRNAP-Dead strain at 37 degrees……………………………………………………………………………………...…….64 Figure 4.14 Substrate consumption and biomass accumulation of the pRNAP-TS strain at 37 degrees………………………………………………………………………………...………….65 Figure 4.15 Substrate consumption and biomass accumulation of the pRNAP-Dead strain at 18 degrees……………………………………………………………………………………………65 Figure 4.16 Substrate consumption, biomass accumulation,